Low heat tolerance and high desiccation resistance in nocturnal bees and the implications for nocturnal pollination under climate change

Predicting insect responses to climate change is essential for preserving ecosystem services and biodiversity. Due to high daytime temperatures and low humidity levels, nocturnal insects are expected to have lower heat and desiccation tolerance compared to diurnal species. We estimated the lower (CTMin) and upper (CTMax) thermal limits of Megalopta, a group of neotropical, forest-dwelling bees. We calculated warming tolerance (WT) as a metric to assess vulnerability to global warming and measured survival rates during simulated heatwaves and desiccation stress events. We also assessed the impact of body size and reproductive status (ovary area) on bees’ thermal limits. Megalopta displayed lower CTMin, CTMax, and WTs than diurnal bees (stingless bees, orchid bees, and carpenter bees), but exhibited similar mortality during simulated heatwave and higher desiccation tolerance. CTMin increased with increasing body size across all bees but decreased with increasing body size and ovary area in Megalopta, suggesting a reproductive cost or differences in thermal environments. CTMax did not increase with increasing body size or ovary area. These results indicate a greater sensitivity of Megalopta to temperature than humidity and reinforce the idea that nocturnal insects are thermally constrained, which might threaten pollination services in nocturnal contexts during global warming.

Table 1S.Results of pairwise comparisons with Bonferroni adjustment of the critical thermal minima (CTMin) and maxima (CTMax) among focal species.Only significant (P < 0.05) comparisons of the 136 total tests for each thermal trait are listed.Table 2S.Critical thermal minima (CTMin) and maxima (CTMax), intertegular distance (ITD), head width (HW), and average ovary size (mm 2 ) among females of Megalopta with different reproductive status.Mean value is followed by SE and sample size.

Figure 1S .
Figure 1S.Temperature (a) and relative air humidity at the forest understory where nests of Megalopta are commonly found (~1 m above ground).Box plots show median, quartiles, and extreme values.For each figure, a different letter above bars indicates significant differences (P < 0.05).

Figure 2S .
Figure 2S.Ambient and internal nest temperature of nocturnal sweat bees of the genus Megalopta.a) Box plots showing median, quartiles, and extreme values of temperatures.Groups with different letters are significantly different (P < 0.05).b) Changes in temperature at 5 min intervals during a 4 h period, from 9:30 to 13:30 h.

Figure 3S .
Figure 3S.Box plots showing CTMin and CTMax among species of diurnal and nocturnal bees.Species are organized into broader taxonomic groups (carpenter bees, orchid bees, stingless bees, and nocturnal bees).For each plot, a different capital letter above taxonomic group indicates significant differences (P < 0.05).To facilitate comparisons, a horizontal line was placed at 10 °C in the CTMin plot and at 45 °C in the CTMax plot.

Figure 4S .
Figure 4S.Critical thermal minima (CTMin) and maxima (CTMax) and their relationship with maximum head width (HW) and average ovary area in nocturnal bees.

Figure 5S .
Figure 5S.Critical thermal minima (CTMin) and maxima (CTMax) among females of Megalopta with different reproductive status.Box plots show median, quartiles, and extreme values of temperatures.For each trait, groups with different letters are significantly different (P < 0.05).

Figure 6S .
Figure 6S.Relationship between survival time of bees exposed to a desiccant and intertegular distance (ITD).

Fig 7S .
Fig 7S.Phylogenetic reconstruction of focal species.Node support indicated by the SH-aLRT value followed by the ultrafast bootstrap value after 1,000 replicates.

Figure 9S .
Figure 9S.Temperature and humidity sensor used in the field, thermal equipment, and desiccation a = Thermochron Fob (red plastic holder) and iButton sensor.b = Plastic holder and iButton shielded with a piece of aluminum foil (~12 ˟ 7 cm).c = Elara 2.0, a portable fully programmable heating/cooling anodized aluminum stage (outlined in red) designed for precision temperature control.Insert in upper right corner shows testing vials with bees, which are plugged with a moisten cotton ball.d = desiccation apparatus filled with fully dehydrated Drierite desiccant (treatment, left tubes) or with a moistened paper towel (control, right tubes).

Table 3S .
Cox proportional hazards estimates of the survival of nocturnal and diurnal bees after exposure to a heat stress event (38 ˚C) over 5 hours.P-values refer to comparisons with nocturnal bees.Significant values in boldface.HR = Hazard ratio; CI = Confidence interval.See Table7Sfor species and sample size used

Table 5S .
Survival time (hour) and percentage of water loss between nocturnal and diurnal bees exposed to a desiccant.Mean value is followed by SE and sample size.See Table7Sfor species and sample size used in this experiment.
74, N = 30 21.88 ±2.63, N = 23 Desiccant 28.04 ±2.56, N = 30 22.35 ±1.77, N = 24 Table 6S.Results of pairwise comparisons with Bonferroni adjustment of the survival time and percentage of water loss of nocturnal and diurnal bees exposed to a desiccant.DF 1 in all comparisons.Significant P-value in boldface.See Table 7S for species and sample size used in this experiment.

Table 7S .
Bee species used in the acute heat stress and desiccation stress assays (indicated with a plus sign).Number of individuals indicated in parentheses.

Table 8S .
GenBank accession numbers for sequences used in this study.